Hello,
perhaps someone is able to help me. I am working on a
ADP-ribosyltransferase. This enzyme transfers the ADP moiety of NAD to
the target enzyme. The determination of the activity works by using
radioactive labeled NAD.
The method is described in Rohrer, H.; Zillig,W. and R. Mailhammer
(1975), Eur. J. Biochem. 60/ 227-238.
The radioactive label is transferred to the target and the amount of
labeled target is determine in a scintilator.
The reaction is stopped by adding 25 µl 50% TCA to the 100 µl
incubation mixture.
The mixture should be transfered to an Nitrocellulose filter
and the filter must be washed to remove the unbound labeled NAD.
And this is the problem. Controls with no ribosyltransferase have as
much cpm´s as the tests with ribosyltransferase
The problem is how to remove the non bound radioactive NAD.
I tried the following methods:
· Transferring the mixtures onto the Filters and washing with 10% TCA
by vacuum
· Transferring the mixtures onto the Filters and washing with Tris
buffer by vacuum
· Transferring the mixture to the Filter with a pipet an dry them at
RT, washing the Filter in a tube with 10 ml binding buffer from
blotting method
· Centrifuge the participated Proteins, and wash them in 500 ml
Aceton. Dissolve the pellet in water and apply them directly to liquid
scintilation.
· Centrifuge the participated Proteins, dissolve them in water and
participate them again by adding TCA and Centrifugation. The pellet is
dissolved again in water an transferred directly to liquid
scintilation.
Perhaps someone has got an idea how to modify the test, to remove the
unbound radioactivity.
Thanks for help,
with best regards
Heiko Koch
Heiko.Koch at ruhr-uni-bochum.de